Field of Invention
The present invention relates to fabrication of porous polymer monoliths and integration of one or more porous polymer monoliths into a fluidic chip. More specifically, embodiments of the present invention relate to ex situ fabrication of porous polymer monoliths and integration of one or more porous polymer monoliths into one or more channels of a channel substrate of a fluidic chip.
Discussion of the Background
Polymer monoliths are a diverse class of porous materials that can be synthesized using a wide variety of monomers, crosslinkers, and polymerization techniques. See, e.g., F. Svec, Porous polymer monoliths: amazingly wide variety of techniques enabling their preparation, Journal of chromatography. A, vol. 1217, no. 6, pp. 902-24, February 2010. With pore sizes that can be tuned from the scale of hundreds of nanometers to tens of microns, polymer monoliths offer a ready alternative to packed particle beds and have been investigated for preparative and analytical applications where high surface area, controllable pore size, and adaptable surface chemistries are advantageous.
Polymer monoliths have been employed in microfluidic systems in a variety of roles. See, e.g., M. Vázquez and B. Paull, Review on recent and advanced applications of monoliths and related porous polymer gels in micro-fluidic devices, Analytica chimica acta, vol. 668, no. 2, pp. 100-13, June 2010. Demonstrated microfluidic applications of porous monoliths include their use as frits for bead packing, cell lysis elements, support scaffolding for micro- and nanoparticles, three dimensional surfaces for antibody and enzyme immobilization, and stationary phases for chromatography or solid phase extraction. See, e.g., S. Zeng et al., Electroosmotic flow pumps with polymer frits, vol. 82, pp. 209-212, 2002; M. Mahalanabis, H. Al-Muayad, M. D. Kulinski, D. Altman, and C. M. Klapperich, Cell lysis and DNA extraction of gram positive and gram-negative bacteria from whole blood in a disposable microfluidic chip, Lab on a chip, vol. 9, no. 19, pp. 2811-7, October 2009; J. Liu, I. White, and D. L. DeVoe, Nanoparticle-functionalized porous polymer monolith detection elements for surface-enhanced Raman scattering, Analytical chemistry, vol. 83, no. 6, pp. 2119-24, March 2011; J. Liu, C.-F. Chen, C.-W. Chang, and D. L. DeVoe, Flow-through immunosensors using antibody-immobilized polymer monoliths, Biosensors & bioelectronics, vol. 26, no. 1, pp. 182-8, September 2010; D. S. Peterson, T. Rohr, F. Svec, and J. M. J. Fréchet, Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices, Analytical chemistry, vol. 74, no. 16, pp. 4081-8, August 2002; J. Liu, C.-F. Chen, C.-W. Tsao, C.-C. Chang, C.-C. Chu, and D. L. DeVoe, Polymer Microchips Integrating Solid-Phase Extraction and High-Performance Liquid Chromatography Using Reversed-Phase Polymethacrylate Monoliths, Analytical Chemistry, vol. 81, no. 7, pp. 2545-2554, April 2009.
Monolith polymerization reactions take place within a solvent solution where individual precursor species exhibit higher solubility than polymerized reaction products. This differential solubility can be controlled by solvent choice or by tuning the solvent: precursor ratio. These parameters may be selected to adjust the rate at which newly polymerized reaction products phase separate and form solid interconnected globules, thereby providing control over morphology of the resulting monolith.
In conventional microfluidic applications, polymer monoliths are formed in situ at the desired locations within a fully assembled microfluidic chip by ultraviolet (UV) photopolymerization of a precursor solution injected into the channels, allowing photolithographic control over the final monolith dimensions. However, in situ integration presents a number of challenges that limit the potential for monoliths in microfluidic applications.
For example, because the optical mask used during contact photolithography is necessarily displaced from the embedded microchannels by the thickness of the microfluidic cover plate, diffraction of light at the mask edges leads to significant variability in UV dose at the boundaries of the exposed region, resulting in poor control over the resolution of the resulting monoliths. In addition, diffusive transport of prepolymer components during the phase separation and polymerization process results in poor monolith homogeneity at the UV-exposed boundary. In particular, pore size and monolith density may differ drastically at the edges. See, e.g., M. He, J.-B. Bao, Y. Zeng, and D. J. Harrison, Parameters governing reproducibility of flow properties of porous monoliths photopatterned within microfluidic channels, Electrophoresis, vol. 31, no. 14, pp. 2422-8, July 2010. The different pore size and monolith density at the edges affect monolith performance for applications such as separations, biosensing, and filtration where uniform pore morphology is critical.
Another constraint that limits the potential of in situ fabricated porous monolith materials for microfluidic applications is that the in situ photopolymerization process requires a solution of monolith precursors, photoinitiators, and porogens to be injected into the microchannels prior to UV exposure, followed by extensive washing steps and any applicable functionalization operations needed to modify the monolith surface. These steps can be cumbersome and highly time consuming, with a typical device requiring a sequence of 3 or more perfusion steps performed over a period of several days. The incursion of precursor, wash, and functionalization solutions into other regions of the microfluidic system during the various perfusion steps can also affect the channel surface chemistry in unintended or undesirable ways.
In situ photopolymerization also necessitates the use of a UV-transparent chip material to allow for exposure through the top or bottom of the chip, limiting the range of substrate materials in which polymer monoliths can be fabricated in situ. Additionally, the chip substrate must also be compatible with the solvent used to induce phase separation during polymerization, further limiting the substrate material options.
Another limitation of in situ fabricated monoliths relates to the attachment of the porous material to the microchannel walls. Because polymer monoliths shrink both during photopolymerization and after aging, chemical attachment methods specific to the channel sidewall material must be implemented to minimize delamination from the channel walls. There are a variety of known attachment schemes. See, e.g., T. B. Stachowiak et al., Fabrication of porous polymer monoliths covalently attached to the walls of channels in plastic microdevices, pp. 3689-3693, 2003; I. Nischang et al., Advances in the preparation of porous polymer monoliths in capillaries and microfluidic chips with focus on morphological aspects, Analytical and bioanalytical chemistry, vol. 397, no. 3, pp. 953-60, June 2010. However, despite the various attachment schemes, avoiding delamination remains a challenge for monoliths fabricated in situ in microchannels. Because monoliths shrink in proportion to their size during polymerization, these attachment schemes become increasingly difficult in larger channels. This phenomenon, combined with the spatial limitations of masked photoinitiation, means that well sealed in situ fabricated monoliths with high aspect ratio (hydrodynamic diameter to length) are exceedingly difficult to produce. Such high aspect ratio monoliths are desirable in applications where a high flow rate or low pressure are required.
A known way of sidestepping some of the constraints of in situ integration of polymer monoliths in channels of a fluidic chip is to integrate capillary-encased monolith segments into a polydimethylsiloxane (PDMS) chip. See, e.g., H. Hisamoto et al., Integration of valving and sensing on a capillary-assembled microchip, Analytical chemistry, vol. 77, no. 7, pp. 2266-71, April 2005. In this case, glass capillaries with rectangular cross-section are used to provide structural integrity for the monolith during insertion into a channel of a fluidic chip. This hybrid glass/elastomer approach allows for covalent attachment between the glass and monolith surfaces, but monolith attachment to the inner wall of the capillary requires additional surface treatment steps. Moreover, the overall capillary integration process imposes a mismatch in cross-sectional dimensions between the channels in the PDMS chip and the capillary-supported monoliths, resulting in a dead volume at the fluidic interfaces between each capillary and the mating channel. In addition, the technique relies on compliant elastomer channel walls for effective leak-free integration of the silica capillaries and, thus, cannot be readily adapted to thermoplastic microfluidic chips fabricated using traditional bonding strategies.
Accordingly, there is a need in the art for improved integration of polymer monoliths into fluidic chips.
There is also a need in the art for improved nucleic acid capture, which is often a necessary step prior to polymerase chain reaction (PCR) amplification during genetic analysis. Nucleic acid capture and purification isolates the nucleic acids from other components of biological sample matrices, such as cell lysate and blood plasma, which could introduce components that inhibit PCR replication of target deoxyribonucleic acid (DNA) sequences, degrade the efficiency of the amplification process, and result in poor assay reproducibility. See, e.g., M. C. Breadmore et al., Microchip-based purification of DNA from biological samples, Anal. Chem., vol. 75, no. 8, pp. 1880-6, April 2003.
Modern laboratory scale DNA purification is typically achieved by silica-based solid phase extraction (SPE) where cell lysate is exposed to a silica surface in the presence of chaotropic agents. See, e.g., N. V. Ivanova et al., An inexpensive, automation-friendly protocol for recovering high-quality DNA, Mol. Ecol. Notes, vol. 6, no. 4, pp. 998-1002, July 2006. This strategy has been employed in a variety of microfluidic formats using packed beds of silica beads and polymer monoliths with embedded silica particles as the solid phase. See, e.g., L. A. Legendre et al., A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples, Anal. Chem., vol. 78, no. 5, pp. 1444-51, March 2006; A. Bhattacharyya and C. M. Klapperich, Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics, Anal. Chem., vol. 78, no. 3, pp. 788-92, February 2006. The extraction efficiency of SPE methods is high (68% to 80%), but the chaotropic agents can be potent PCR inhibitors, thereby requiring copious washing to ensure that an inhibitor-free DNA solution is eluted as a final product.
An aqueous and PCR compatible alternate approach to chaotropic SPE is electrostatically driven, pH modulated nucleic acid capture on an amine-rich surface that can be controllably switched between cationic and neutral states. Such charge switching methods have been implemented in microfluidic systems, with various aminosilanes used to coat glass microchannels to yield a capture substrate with pH switchable surface charge. See, e.g., J. Wen et al., DNA extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase, Anal. Chem., vol. 78, no. 5, pp. 1673-81, March 2006.
As an effective alternative to aminosilanes, the aminosacharide biopolymer chitosan has also been employed as a pH modulated surface treatment for nucleic acid capture in microfluidic devices. See e.g., W. Cao et al., Chitosan as a polymer for pH-induced DNA capture in a totally aqueous system, Anal. Chem., vol. 78, no. 20, pp. 7222-8, October 2006; C. R. Reedy et al., Solid phase extraction of DNA from biological samples in a post-based, high surface area poly(methyl methacrylate) (PMMA) microdevice, Lab Chip, vol. 11, no. 9, pp. 1603-11, May 2011; K. A. Hagan et al., Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device, Anal. Chem., vol. 81, no. 13, pp. 5249-56, July 2009.
While high loading levels and extraction efficiencies have been reported using chitosan as a charge-switching polymer for microfluidic DNA capture and release, known methods require long channels distributed over large device areas to achieve this performance. This constraint is imposed by the need for sufficient surface area to achieve acceptable loading capacity. While high aspect ratio microstructures can be used to enhance surface area, this approach requires the application of complex fabrication methods that are undesirable for use in disposable sample preparation chips. Furthermore, long or wide channels are required so that the residence time during perfusion through the capture zone is significantly longer than the characteristic diffusion time for each sample component, ensuring sufficient interactions between DNA and the channel walls to promote efficient capture.
An approach for the direct attachment of proteins to glycidyl methacrylate (GMA) monoliths is known. See R. Mallik et al., High-Performance Affinity Monolith Chromatography: Development and Evaluation of Human Serum Albumin Columns preparation of high-performance affinity monolithic col-, vol. 76, no. 23, pp. 7013-7022, 2004. In this approach, direct reaction between primary amines of the protein and the exposed reactive epoxy groups on the GMA monolith is achieved in the absence of a separate cross-linking agent, providing a simple and convenient route to monolith functionalization. However, due to the limited reaction efficiency, this technique required a 6 day, room temperature perfusion of protein solution at pH 8 through the GMA monolith to achieve adequate conjugation. For conventional microfluidic applications, where monoliths are fabricated in situ within a microchannel by patterned photopolymerization, the extended reaction time renders the direct reaction route impractical, since each individual chip requires photolithographic fabrication of the monolith elements followed by extended reagent perfusion and incubation.
Accordingly, there is a need in the art for improved nucleic acid capture using chitosan.